Autophagy is an essential lysosomal degradation pathway that removes damaged organelles and protein aggregates from the cell. Autophagy is essential in post-mitotic cells such as neurons since they are unable to dilute out proteotoxins by cell division. In fact, neuron-specific loss of autophagy is sufficient to cause neuron cell death. Further, multiple neurodegenerative diseases characterized by excessive protein aggregation exhibit pronounced defects in autophagy. Despite the clear implications of defective autophagy in disease, little is known about the basic mechanisms driving autophagy in neurons. Preliminary data indicate that autophagosomes are preferentially generated in the distal neurite. Initially, they exhibit bidirectional motility but then exit the distal region and undergo robust retrograde transport to the cell soma. This shift in motility is accompanied by fusion with late endosomes/lysosomes. As autophagosomes travel toward the cell soma, they continue to acidify and mature into autolysosomes that may more effectively degrade cargo. Potentially, delivery of autolysosomes to the cell soma ensures rapid and efficient recycling of degradation products to primary sites of protein synthesis. Based on the preliminary data, this proposal will test the hypothesis that autophagosome biogenesis and maturation are spatially and temporally regulated along the axon of primary neurons. Further, this study will also test the hypothesis that autophagosome function and transport are tightly linked. To examine these hypotheses, this proposal will (1) determine the mechanisms of autophagosome biogenesis in primary neurons (2) determine the relationship between autophagosome transport and maturation in primary neurons under basal versus stress conditions and (3) determine the mechanisms of cargo degradation by autophagy in primary neurons under basal versus stress conditions. Together, this proposal will determine the mechanisms of autophagosome biogenesis from birth to maturation into degradative and functional organelles and how this pathway becomes altered in response to cellular stressors such as mitochondrial damage and protein aggregation. These hypotheses will be tested using a multidisciplinary approach ranging from live-cell imaging to biophysical techniques in neurons isolated from wild type animals and models of neurodegenerative disease. During the K99 phase, new methodologies in neurobiology and biophysics will be established to examine the proposed hypotheses on a mechanistic level. These methodologies will then be utilized during the R00 phase to establish an innovative and independent research program. Results from this study will uncover novel information about the regulation of autophagy in primary neurons and have significant implications on understanding the progression of neurodegenerative disease.